Osteoarthritis (OA) is a common, debilitating condition for which there is no cure and few treatments. Over the last several years, glucosamine has been endorsed in the lay-press as a useful over-the-counter remedy for OA. In controlled clinical studies, however, reports of the efficacy of glucosamine have been inconsistent, leaving skepticism that its ingestion as a dietary supplement mediates a meaningful biological response in the joint tissues. Recently glucosamine and its effects on chondrocytes and cartilage biology have fallen under scrutiny in laboratory studies. Initial in vitro work showed that glucosamine possessed certain chondroprotective properties, moderating aspects of the deleterious response of chondrocytes to inflammatory stimulation. Using microarray analyses, we have recently shown that glucosamine alone had little effect on genes involved in matrix synthesis, but indeed was a highly effective antagonist of interleukin- 1, enabling broad-spectrum inhibition. For example, of the -1600 genes whose transcription was altered by IL-1p stimulation (P<0.0001), glucosamine significantly inhibited the response in -1200. However, in vitro chondroprotection was observed only in the presence of very high pericellular concentrations of glucosamine that far exceed those achievable by standard oral administration. Thus, while glucosamine appears to have important chondroprotective properties, its full therapeutic potential cannot be realized through conventional delivery. This proposal is based upon a novel, gene-based method of increasing the endogenous, intra- articular synthesis of glucosamine that, unlike oral administration, will sustain high, potentially therapeutic levels in the joint. We hypothesize that synovial fibroblasts genetically modified to overexpress glutamine: fructose 6 amido transl'erase (GFAT) willproduce large amounts of glucosamine intra-articlularly sufficient to provide effective chondroprotection in OA. To test this hypothesis, the following specific aims will be addressed: 1). To determine global transcriptional patterns in chondrocvtes and svnoviocvtes treated by glucosamine or GFAT gene transfer following IL-1B challenge. Using a new co-culture system and microarray analyses we will characterize both the nature and extent of chondroprotection afforded by GFAT overexpression in synovial fibroblasts. Resulting transcriptional profiles will be compared to those obtained from the addition of exogenous glucosamine. 2). To evaluate the ability of long term expression of GFAT in svnovial cells to protect against cartilage degradation in an experimental model of OA. The rat anterior cruciate ligament transection model of OA will be used to determine if lentiviral-mediated delivery of GFAT in the synovial lining confers chondroprotection in the articular cartilage. Results will be compared with those obtained using ACL-transected animals daily fed with glucosamine. If successfully completed, this study will lead to further insight to the biological effects of glucosamine on arthritis and perhaps the development of a novel chondroprotective strategy for the treatment of OA.